Cell permeable peptides for inhibition of inflammatory reactions and methods of use

ABSTRACT

The present invention relates to the delivery of biologically active molecules, such as peptides, into the interior of cells by administering to the cells a complex comprising the molecule linked to an importation competent signal peptide. Such delivery can be utilized, for example, to treat and/or prevent inflammatory conditions, e.g., but not limited to, systemic inflammatory reactions such as endotoxic shock, localized inflammatory reactions such as inflammatory skin diseases and conditions, and inflammatory diseases such as autoimmune diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 national phase applicationfiled from, and claiming priority to, international applicationPCT/US00/32516, filed Nov. 29, 2000 (published under PCT Article 21(2)in English), which is a continuation-in-part of U.S. Ser. No.09/450,071, filed Nov. 29, 1999 now U.S. Pat. No. 6,495,518 (nowallowed), which is a continuation-in-part of U.S. Ser. No. 09/170,754,filed Oct. 13, 1998 (now U.S. Pat. No. 6,043,339), which is acontinuation of U.S. Ser. No. 09/052,784, filed Mar. 31, 1998 (nowabandoned), which is a continuation of U.S. Ser. No. 08/258,852, filedon Jun. 13, 1994 (now U.S. Pat. No. 5,807,746), which applications areherein incorporated by reference in their entireties.

ACKNOWLEDGMENTS

This invention was made with partial government support under NIH GrantNos. HL45994, H62356, and DK54072. The United States government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to biologically active molecules and tomethods for delivery of biologically active molecules into the interiorof cells by administering to the cells a complex comprising the moleculelinked to a signal peptide. The present invention also relates to thedevelopment of cell-permeable peptide analogs and to methods for thetargeted delivery of these peptide analogs to control systemicinflammatory response syndromes such as endotoxic shock, as well as abroad variety of inflammatory diseases and conditions.

BACKGROUND OF THE INVENTION

Peptides have been developed for many therapeutic uses. For example,diseases currently targeted by new peptide drugs include heartconditions, cancers, endocrine disorders, neurological defects,respiratory conditions, allergies and autoimmune diseases. Although themanufacture of known therapeutic peptides can be achieved by knownmethods, i.e., classic synthetic techniques or recombinant geneticengineering, delivery of the peptides into a cell has remainedproblematic, since they cannot readily cross biological membranes toenter cells. Thus, current methods include permeabilization of the cellmembrane, or microinjection into the cell. Both of these methods haveserious drawbacks. Permeabilization of cells, e.g., by saponin,bacterial toxins, calcium phosphate, electroporation, etc., can only bepractically useful for ex vivo methods, and these methods cause damageto the cells. Microinjection requires highly skilled technicians (thuslimiting its use to a laboratory setting), it physically damages thecells, and it has only limited applications as it cannot be used totreat for example, a mass of cells or an entire tissue, because onecannot feasibly inject large numbers of cells.

Similarly, delivery of nucleic acids has been problematic. Methodscurrently employed include the permeabilization described above, withthe above-described drawbacks, as well as vector-based delivery, such aswith viral vectors, and liposome-mediated delivery. However, viralvectors can present additional risks to a patient, and liposometechniques have not achieved satisfactorily high levels of delivery intocells.

Signal peptide sequences, ¹ which share the common motif ofhydrophobicity, mediate translocation of most intracellular secretoryproteins across mammalian endoplasmic reticulum (ER) and prokaryoticplasma membranes through the putative protein-conducting channels.²⁻¹¹Alternative models for secretory protein transport also support a rolefor the signal sequence in targeting proteins to membranes.¹²⁻¹⁵

Several types of signal sequence-mediated inside-out membranetranslocation pathways have been proposed. The major model implies thatthe proteins are transported across membranes through a hydrophilicprotein conducting channel formed by a number of membrane proteins.²⁻¹¹In eukaryotes, newly synthesized proteins in the cytoplasm are targetedto the ER membrane by signal sequences that are recognized generally bythe signal recognition particle (SRP) and its ER membrane receptors.This targeting step is followed by the actual transfer of protein acrossthe ER membrane and out of the cell through the putativeprotein-conducting channel (for recent reviews, see references 2-5). Inbacteria, the transport of most proteins across the cytoplasmic membranealso requires a similar protein-conducting channel.⁷⁻¹¹ On the otherhand, signal peptides can interact strongly with lipids, supporting theproposal that the transport of some secretory proteins across cellularmembranes may occur directly through the lipid bilayer in the absence ofany proteinaceous channels.¹⁴⁻¹⁵

Thus, though many attempts have been made to develop effective methodsfor importing biologically active molecules into cells, both in vivo andin vitro, none has proved to be entirely satisfactory.

SUMMARY OF THE INVENTION

The present invention provides methods and peptides for treatinginflammatory diseases and conditions.

For example, the invention provides a method for treating or preventingan inflammatory response in a subject. The method includes administeringto the subject a peptide containing an NF-kB nuclear localizationsequence such that nuclear import of a stress-responsive transcriptionfactor is inhibited in a cell of the subject, thereby treating orpreventing an inflammatory response in the subject.

The invention also provides a method for treating or preventing septicshock in a subject, including delivering to the subject a compoundincluding a peptide including a nuclear localization sequence of NF-kBsuch that nuclear import of NF-kB is inhibited, thereby treating orpreventing septic shock in the subject.

The present invention further provides a method of importing abiologically active molecule into a cell in a subject comprisingadministering to the subject a complex comprising the molecule linked toan importation competent signal peptide, thereby importing the moleculeinto the cell of the subject.

Additionally, the instant invention provides a method of importing abiologically active molecule into the nucleus of a cell in a subjectcomprising administering to the subject a complex comprising themolecule linked to an importation competent signal peptide and a nuclearlocalization peptide, thereby importing the molecule into the nucleus ofthe cell of the subject.

The present invention also provides a complex comprising an importationcompetent signal peptide linked to a biologically active moleculeselected from the group consisting of a nucleic acid, a carbohydrate, alipid, a glycolipid and a therapeutic agent.

The invention also provides peptides for use in the methods of theinvention, such as a peptide including the amino acid sequence set forthin SEQ ID NO: 9; a peptide including the amino acid sequence set forthin SEQ ID NO: 12; and a peptide including the amino acid sequence setforth in SEQ ID NO:13.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and upon payment of thenecessary fee.

FIG. 1 is a graph showing [³H] thymidine incorporation by NIH 3T3 cellsstimulated with either (a) SA peptide, SAα peptide, ANL peptide or SMpeptide or (b) acidic Fibroblast Growth Factor (aFGF).

FIGS. 2A-2D is a series of graphs demonstrating the improved survival ofC57Bl/6 mice treated with the SN50 peptide as compared to untreated orSM-peptide-treated controls. The groups of 5 mice each receivedintraperitoneal injections of D-galactosamine (20 mg in pyrogen-freesaline) without or with peptide (2 mg) 30 min. before LPS from E. coli0127:B8. The peptide injections were repeated at 30, 90, 150, and 210minutes following LPS (as shown in FIGS. 2B and 2C); additional twoinjections were administered at 6 and 12 h following LPS (FIG. 2D).Surviving mice were euthanized after 72 h. Cumulative results of 2-3groups are presented in FIG. 3A (control mice treated with 5 injectionof saline (diluent)); FIG. 2B (animals treated with 5 injections of SMpeptide); FIG. 2C (animals treated with 5 injections of SN50 peptide);and, FIG. 2D (animals treated with 7 injections of SN50 peptide).

FIGS. 3A-3E is a series of graphs illustrating the survival of miceafter LPS injection. Female C57Bl/6 mice(20 g) were randomly grouped (5mice per group) and received intraperitoneal injections of LPS (E. coli0127:B5, 800 μg). Treatments included cSN50 (1.5 mg or 0.7 mg) and SMpeptide (1.5 mg) given 30 min before LPS, and afterwards at 30, 90, 150,210 minutes and 6 hrs and 12 hrs. FIG. 3A shows the survival rate wherethe control (saline) was used. FIG. 3B illustrates that rate where cSN50peptide was administered at 0.7 mg×7. FIG. 3C shows the survival ratewhere cSN50 peptide was administered at 1.5 mg×7. FIG. 3D illustratesthat rate where SM peptide was administered at 1.5 mg×7. FIG. 3E showsthe survival rate where cSN50 peptide (1.5 mg) was administered 30 minafter endotoxin followed by 0.7 mg injections at 90, 150, 210 min and 6,12, and 24 hrs.

FIG. 4 is a diagram of an electrophoretic mobility shift assay (EMSA)showing the inhibitory effect of the cSN50 peptide of NF-κB nuclearimport in T cells.

FIG. 5 is a graph showing that death from staphylococcal enterotoxin B(SEB)-induced toxic shock in mice is prevented by inhibition of NF-κBnuclear import by the NF-κB NLS-containing peptide cSN50.

FIGS. 6A-6D are diagrams showing photomicrographs of liver sections,stained with either hematoxylin and eosin (H & E) or Apop Tag, fromuntreated SEB-challenged mice and SEB-challenged mice treated with theNF-κB NLS-containing peptide cSN50.

FIG. 7 shows that both the L- and D- isomers of Membrane PermeableSequence (MPS) were able to deliver nuclear localization sequence (NLS)to the cytoplasm of murine endothelial LE II cells (FIG. 7A) and humanerythroleukemia cells (FIG. 7B), as evidenced by concentration dependentinhibition of nuclear import of NF-kB induced by pro-inflammatoryagonists LPS (FIG. 7A) and TNF-α(FIG. 7B). Thus, intracellular deliveryof functional peptides is not dependent on chirality of MPS, indicatingthat a specific receptor or transporter protein is not involved.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesand Figures included herein.

The present invention provides the discovery that importing exogenousbiologically active molecules into intact cells can be engineered byforming a complex by attaching an importation competent signal peptidesequence (also referred to by the synonymous term “cellmembrane-permeable hydrophobic region of a signal peptide”) to aselected biologically active molecule and administering the complex tothe cell. The complex is then imported across the cell membrane by thecell. Thus, the present invention provides a method of importing abiologically active molecule into a cell ex vivo or in vivo comprisingadministering to the cell, under import conditions, a complex comprisingthe molecule linked to an importation competent signal peptide (alsoknown as a cell membrane-permeable hydrophobic region of a signalpeptide), thereby importing the molecule into the cell.

As used herein, “biologically active molecule” includes any moleculewhich if imported into a cell, can have a biological effect. Naturallyonly those molecules which are of a size which can be imported into thecell are within the scope of the invention. However, since very largeproteins (ranging from molecular weights of about 100,000 to around 1million) are exported by cells (e.g., antibodies, fibrinogen, andmacroglobulin), very large proteins can be imported into cells by thismethod. Therefore, size ranges for proteins from a few amino acids toaround a thousand amino acids can be used. A preferable size range forproteins is from a few amino acids to about 250 amino acids. For anymolecule, size ranges can be up to about a molecular weight of about 1million, with a preferable size range being up to a molecular weight ofabout 25,000, and an even more preferable size range being up to amolecular weight of about 3,000. In addition, only those molecules whichcan be linked to a signal peptide, either directly or indirectly, arewithin the scope of the invention. Likewise, the present inventionrequires that the complex is a administered under suitable conditionsfor effective import into the cell.

Examples of biologically active molecules include proteins, polypeptidesand peptides, which include functional domains of biologically activemolecules, such as growth factors, enzymes, transcription factors,toxins, antigenic peptides (as for vaccines), antibodies, and antibodyfragments. Additional examples of biologically active molecules includenucleic acids, such as plasmids, coding DNA sequences, mRNAs andantisense RNA molecules, carbohydrates, lipids and glycolipids. Furtherexamples of biologically active molecules include therapeutic agents, inparticular those with a low cell membrane permeability. Some examples ofthese therapeutic agents include cancer drugs, such as Daunorubicin,²⁶and toxic chemicals which, because of the lower dosage that can beadministered by this method, can now be more safely administered.

A specific example of a biologically active molecule is the peptidecomprising the nuclear location sequence (NLS) of acidic fibroblastgrowth factor (aFGF), listed herein as SEQ ID NO:2. As demonstrated inthe examples below, the NLS of aFGF, when linked to a signal peptide andtransported into cells (e.g., the entire peptide listed herein as SEQ IDNO:4), induces a rnitogenic response in the cells. Another example of abiologically active molecule is the peptide comprising the NLS oftranscription factor NF-kB subunit p50, listed herein as SEQ ID NO:10.As shown in the examples herein, when a peptide comprising the signalsequence of K-FGF and the NLS of transcription factor NF-kB p50 subunit,this peptide (called SN50) being listed herein as SEQ ID NO:9, istransfected into cells having transcription factor NF-kB, the normaltranslocation of active NF-kB complex into the nucleus is inhibited. Inthis manner, cell growth can be inhibited by inhibiting the action ofNF-kB and therefore inhibiting the expression of genes controlled bytranscription factor NF-kB.

Yet another example of a biologically active molecule is an antigenicpeptide. Antigenic peptides can be administered to provide immunologicalprotection when imported by cells involved in the immune response. Otherexamples include immunosuppressive peptides (e.g., peptides that blockautoreactive T cells, which peptides are known in the art). Numerousother examples will be apparent to the skilled artisan.

Suitable import conditions are exemplified herein and include cell andcomplex temperature between about 180° C. and about 42° C., with apreferred temperature being between about 22° C. and about 37° C. Foradministration to a cell in a subject the complex, once in the subject,will of course adjust to the subject's body temperature. For ex vivoadministration, the complex can be administered by any standard methodsthat would maintain viability of the cells, such as by adding it toculture medium (appropriate for the target cells) and adding this mediumdirectly to the cells. As is known in the. art, any medium used in thismethod can be aqueous and non-toxic so as not to render the cellsnon-viable. In addition, it can contain standard nutrients formaintaining viability of cells, if desired. For in vivo administration,the complex can be added to, for example, a blood sample or a tissuesample from the patient or to a pharmaceutically acceptable carrier,e.g., saline and buffered saline, and administered by any of severalmeans known in the art. Examples of administration include parenteraladministration, e.g., by intravenous injection including regionalperfusion. through a blood vessel supplying the tissues(s) or organ(s)having the target cell(s), or by inhalation of an aerosol, subcutaneousor intramuscular injection, topical administration such as to skinwounds and 1 lesions, direct transfection into, e.g., bone marrow cellsprepared for transplantation and subsequent transplantation into thesubject, and direct transfection into an organ that is subsequentlytransplanted into the subject. Further administration methods includeoral administration, particularly when the complex is encapsulated, orrectal administration, particularly when the complex is in suppositoryform. A pharmaceutically acceptable carrier includes any material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected complex withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is administered. Administrationcan be performed for a time length of about 1 minute to about 72 hours.Preferable time lengths are about 5 minutes to about 48 hours, and evenmore preferably about 5 minutes to about 20 hours, and even morepreferably about 5 minutes to about 2 hours. Optimal time lengths andconditions for any specific complex and any specific target cell canreadily be determined, given the teachings herein and knowledge in theart. ²⁷ Specifically, if a particular cell type in vivo is to betargeted, for example, by regional perfusion of an organ or tumor, cellsfrom the target tissue can be biopsied and optimal dosages for import ofthe complex into that tissue can be determined in vitro, as describedherein and as known in the art, to optimize the in vivo dosage,including concentration and time length. Alternatively, culture cells ofthe same cell type can also be used to optimize the dosage for thetarget cells in vivo.

For either ex vivo or in vivo use, the complex can be administered atany effective concentration. An effective concentration is that amountthat results in importation of the biologically active molecule into thecell. Such a concentration will typically be between about 0.5 nM toabout 100 μM (culture medium concentration (ex vivo) or blood serumconcentration (in vivo)). Optimal concentrations for a particularcomplex and/or a particular target cell can be readily determinedfollowing the teachings herein. Thus, in vivo dosages of the complexinclude those which will cause the blood serum concentration of thecomplex to be about 0.5 nM to about 100 μM. A preferable concentrationis about 2 nM to about 50 μM. The amount of the complex administeredwill, of course, depend upon the subject being treated, the subject'sage and weight, the manner of administration, and the judgment of theskilled administrator. The exact amount of the complex will furtherdepend upon the general condition of the subject, the severity of thedisease/condition being treated by the administration and the particularcomplex chosen. However, an appropriate amount can be determined by oneof ordinary skill in the art using routine optimization given theteachings herein.

Parenteral administration, e.g., regional perfusion, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, such as liquid solutions, suspensions, or emulsions.A slow release or sustained release system, such as disclosed in U.S.Pat. No. 3,710,795, can also be used, allowing the maintenance of aconstant level of dosage.

Depending on the intended mode of administration (e.g., but not limitedto, intravenous, parenteral, transcutaneous, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal,intrarectal, intravaginal, aerosol, or oral), the pharmaceuticalcompositions may be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, lotions, creams, gels, or the like,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include, as noted above, aneffective amount of the selected drug in combination with apharmaceutically acceptable carrier and, in addition, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,etc.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. an active compound as described herein, and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the Eke. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences. ²⁷

The present invention utilizes a complex comprising the selectedbiologically active molecule linked to an importation competent signalpeptide or cell membrane-permeable hydrophobic region of a signalpeptide. As discussed above, the biologically active molecule can beselected from any of a variety of molecules, with its selection beingdependent upon the purpose to be accomplished by importing the moleculeinto the selected cell. An “importation competent signal peptide” or“cell membrane-permeable hydrophobic region of a signal peptide” as usedherein, is a sequence of amino acids generally of a length of about 10to about 50 or more amino acid residues, many (typically about 55-60%)residues of which are hydrophobic such that they have a hydrophobic,lipid-soluble portion. ¹ The hydrophobic portion is a common, majormotif of the signal peptide, and it is often a central part of thesignal peptide of protein secreted from cells. A signal peptide is apeptide capable of penetrating through the cell membrane to allow theexport of cellular proteins. The signal peptides of this invention, asdiscovered herein, are also “importation competent” or “cell-permeable,”i.e., capable of penetrating through the cell membrane from outside thecell to the interior of the cell. The amino acid residues can be mutatedand/or modified (i.e., to form mimetics) so long as the modifications donot affect the translocation-mediating function of the peptide. Thus theword “peptide” includes mimetics and the word “amino acid” includesmodified amino acids, as used herein, unusual amino acids, and D-formamino acids. All importation competent signal peptides encompassed bythis invention have the function of mediating translocation across acell membrane from outside the cell to the interior of the cell. Suchimportation competent signal peptides could potentially be modified suchthat they lose the ability to export a protein but maintain the abilityto import molecules into the cell. A putative signal peptide can easilybe tested for this importation activity following the teachings providedherein, including testing for specificity for any selected cell type.

Signal peptides can be selected, for example, from the SIGPEP database,which also lists the origin of the signal peptide. ^(30,38) When aspecific cell type is to be targeted, a signal peptide used by that celltype can be chosen. For example, signal peptides encoded by a particularoncogene can be selected for use in targeting cells in which theoncogene is expressed. Additionally, signal peptides endogenous to thecell type can be chosen for importing biologically active molecules intothat cell type. And again, any selected signal peptide can be routinelytested for the ability to translocate across the cell membrane of anygiven cell type according to the teachings herein. Specifically, thesignal peptide of choice can be conjugated to a biologically activemolecule, e.g., a functional domain of a cellular protein or a reporterconstruct, and administered to a cell, and the cell is subsequentlyscreened for the presence of the active molecule. The presence ofmodified amino acids in the signal peptide can additionally be usefulfor rendering a complex, wherein the biologically active molecule is apeptide, polypeptide or protein, more resistant to peptidase in thesubject. Thus these signal peptides can allow for more effectivetreatment by allowing more peptides to reach their target and byprolonging the life of the peptide before it is degraded. Additionally,one can modify the amino acid sequence of the signal peptide to alterany proteolytic cleavage site present in the original signal sequencefor removing the signal sequence. Clearage sites are characterized bysmall, positively charged amino acids with no side chains and arelocalized within about 1 to about 4 amino acids from the carboxy end ofthe signal peptide. ¹

An example of a useful signal peptide is the signal peptide from Capassofibroblast growth factor (K-FGF),¹⁶⁻¹⁷ listed herein as SEQ ID NO:5. Anysignal peptide, however, capable of translocating across the cellmembrane into the interior of the selected target cell can be usedaccording to this invention.

By “linked” as used herein is meant that the biologically activemolecule is associated with the signal peptide in such a manner thatwhen the signal peptide crosses the cell membrane, the molecule is alsoimported across the cell membrane. Examples of such means of linkinginclude (1) when the molecule is a peptide, the signal peptide (and anuclear localization peptide, if desired) can be linked by a peptidebond, i.e., the two peptides can be synthesized contiguously; (2) whenthe molecule is a polypeptide or a protein (including antibody), thesignal peptide (and a nuclear localization peptide, if desired), can belinked to the molecule by a peptide bond or by a non-peptide covalentbond (such as conjugating a signal peptide to a protein with across-linking reagent); (3) for molecules that have a negative charge,such as nucleic acids, the molecule, and the signal peptide (and anuclear localization peptide, if desired) can be joined bycharge-association between the negatively charged molecule and thepositively-charged amino acids in the peptide or by other types ofassociation between nucleic acids and amino acids; (4) chemical ligationmethods can be employed to create a covalent bond between thecarboxy-terminal amino acid of the signal peptide (and a nuclearlocalization peptide, if desired) and the molecule. Methods (1) and (2)are typically preferred.

Examples of method (1) are shown below wherein a peptide is synthesized,by standard means known in the art, ^(24,25) that contains, in linearorder from the amino-terminal end, a signal peptide sequence, anoptional spacer amino acid region, and a biologically active amino acidsequence. Such a peptide could also be produced through recombinant DNAtechniques, expressed from a recombinant construct encoding theabove-described amino 10 acids to create the peptide. ²⁸

For method (2), either a peptide bond, as above, can be utilized or anon-peptide covalent bond can be used to link the signal peptide withthe biologically active polypeptide or protein. This non-peptidecovalent bond can be formed by methods standard in the art, such as byconjugating the signal peptide to the polypeptide or protein via across-linking reagent, for example, glutaraldehyde. Such methods arestandard in the art. ²⁹ For method (3) the molecules can simply be mixedwith the signal peptide and thus allowed to associate. These methods areperformed in the same manner as association of nucleic acids withcationic liposomes. ³²⁻³⁴ Alternatively, covalent (thioester) bonds canbe formed between nucleic acids and peptides. Such methods are standardin the art.

For method (4), standard chemical ligation methods, such as usingchemical cross-linkers interacting with the carboxy-terminal amino acidof the signal peptide, can be utilized. Such methods are standard in theart (see, e.g., Goodfriend, ³¹ which uses water-soluble carbodfimide asa ligating reagent) and can readily be performed to link the carboxyterminal end of the signal peptide to any selected biologically activemolecule.

The complex that is administered to a subject can further comprise aliposome. Cationic and anionic liposomes are contemplated by thisinvention, as well as liposomes having neutral lipids. Cationicliposomes can be complexed with the signal peptide and anegatively-charged biologically active molecule by mixing thesecomponents and allowing them to charge-associate. Cationic liposomes areparticularly useful when the biologically active molecule is a nucleicacid because of the nucleic acid's negative charge. Examples of cationicliposomes include lipofectin, lipofectamine, lipofectace and DOTAP.³²⁻³⁴ Anionic liposomes generally are utilized to encase within theliposome the substances to be delivered to the cell. Procedures forforming cationic liposomes encasing substances are standard in the art³⁵ and can readily be utilized herein by one of ordinary skill in theart to encase the complex of this invention.

Any selected cell into which import of a biologically active moleculewould be useful can be targeted by this method, as long as there is ameans to bring the complex in contact with the selected cell. Cells canbe within a tissue or organ, for example, supplied by a blood vesselinto which the complex is administered. Additionally, the cell can betargeted by, for example, inhalation of the molecule linked to thepeptide to target the lung epithelium. Some examples of cells that canbe targeted by this inventive method include fibroblasts, epithelialcells, endothelial cells, blood cells and tumor cells, among many. Inaddition, the complex can be administered directly to a tissue site inthe body. As discussed above, the signal peptide utilized can be chosenfrom signal peptides known to be utilized by the selected target cell,or a desired signal peptide can be tested for importing ability giventhe teachings herein. Generally, however, all signal peptides have thecommon ability to cross cell membranes due, at least in part, to theirhydrophobic character. Thus, in general, a membrane-permeable signalpeptide can be designed and used for any cell type, since all eukaryoticcell membranes have a similar lipid bilayer.

One particularly useful example is to import an antigenic peptide intocells of the immune system, thereby allowing the antigen to be presentedby antigen-presenting cells, and an immune response to the antigen to bedeveloped by the subject. These antigenic peptide-containing complexescan be administered to the subject according to standard methods ofadministering vaccines, e.g., intramuscularly, subcutaneously or orally,and effectiveness can be measured by subsequent measuring of thepresence of antibodies to the antigen. The present invention alsoprovides a method of importing a biologically active molecule into thenucleus of a cell in a subject comprising administering to the subject acomplex comprising the molecule linked to an importation competentsignal peptide and a nuclear localization peptide, thereby importing themolecule into the nucleus of the cell of the subject. A nuclearlocalization peptide, as used herein, is a peptide having the functionof delivering an intracellular peptide into the nucleus of the cell.Such nuclear localization sequences are known in the art to have thisfunction ^(36,37). An example of a nuclear localization peptide is thenuclear localization sequence of aFGF, listed herein as SEQ ID NO:2. Anexample of a signal peptide (K-FGF) linked to a nuclear localizationpeptide (aFGF) is set forth in SEQ ID NO:3. As these examplesdemonstrate, the nuclear localization peptide sequences can besynthesized as a peptide contiguous with the signal peptide, if desired.Additionally, separate peptides can be linked by any means such asdescribed herein.

The present invention provides a method for treating or preventingsepsis (septic shock) in a subject, e.g., a human subject, comprisingdelivering to the subject a compound comprising a nuclear localizationsequence of NF-kB such that nuclear importation of NF-kB is inhibited ina presently preferred embodiment, one or all of AP-1, NFAT and STAT-1are also inhibited.

In one embodiment exemplified below, the nuclear localization sequenceof NF-kB is delivered into the cells of the subject by linkage to animportation competent signal peptide. See also, Rojas, M. et al., 1998Nature Biotechnology 16:370-375. However, the nuclear localizationsequence of NF-kB could also be delivered by other means such as byphysical methods of introducing proteins into cells (microinjection,electroporation, biolistics); chemical or biological pore formation(digitonin, pore forming proteins and ATP treatment); use of modifiedproteins (lipidated proteins and bioconjugates, such as with animmunotoxin); and, particle uptake (microspheres, virus mimics, inducedpinocytosis). Patton, J., 1998 Nature Biotechnology 16:141-143; Putneyand Burke, 1998 Nature Biotechnology 16:153-157 and Fernandez andBayley, 1998 Nature Biotechnology 16:418-420.

Alternatively, one could deliver the nuclear localization sequence ofNF-kB by administering to the subject a nucleic acid encoding a nuclearlocalization sequence of NF-kB. Such a nucleic acid could be deliveryfor example as naked DNA, with a viral vector, or by means such ascationic liposomes.

The present invention also provides a method of importing a biologicallyactive molecule into the nucleus of a cell in a subject comprisingadministering to the subject a complex comprising the molecule linked toan importation competent signal peptide and a nuclear localizationpeptide, thereby importing the molecule into the nucleus of the cell ofthe subject.

The present invention also provides a method of regulating growth of acell in a subject comprising administering to the subject a complexcomprising a growth regulatory peptide linked to an importationcompetent signal peptide to import the growth regulatory peptide intothe cell of the subject thereby regulating the growth of the cell.Growth can be stimulated or inhibited depending upon the growthregulatory peptide selected. It is to be noted that the presentinvention provides regulation of cell growth also by administering anucleic acid encoding a growth regulatory peptide under functionalcontrol of a suitable promoter for expression in a specific target cell,wherein the nucleic acid is complexed with a signal peptide andadministered to the target cell.

There are numerous growth regulatory peptides known in the art, any ofwhich can be utilized in this invention, if appropriate for the targetcell type and the type of regulation desired. The signal peptidefacilitates the efficient import of the growth regulatory peptide intothe target cell and, once the regulatory peptide is imported, itfunctions to regulate cell growth in its specific manner. A particularlyuseful target cell is a tumor cell in which the method can be used toinhibit further aberrant cell growth. Cell growth can be stimulated byadministering a growth regulatory peptide comprising the nuclearlocalization sequence of acidic fibroblast growth factor (aFGF). Cellgrowth can be inhibited by administering peptides that inhibit growth,for example peptides that inhibit transcription in the cell, such as theNLS of the p50 subunit of transcription factor NF-kB.

An example of this method is seen below in the examples wherein thegrowth regulatory peptide stimulates cell growth and comprises thenuclear localization signal of aFGF. As this example demonstrates, thegrowth regulatory peptide, if desired, can be synthesized contiguouslywith the signal peptide, though any known method can be utilized to linkthem. An example of a contiguous peptide is set forth in SEQ ID NO:3 andSEQ ID NO:4. Another example is provided below, wherein a complex(listed as SEQ ID NO:9) comprising the membrane-permeable signal peptideof K-FGF linked to the NLS of transcription factor NF-kB p50 subunit isadministered and inhibits the expression of genes encodingpro-inflammatory mediators.

The invention also provides a method of inhibiting expression in a cellin a subject of a gene controlled by transcription factor NF-kBcomprising administering to the subject a complex comprising animportation competent signal peptide linked to a nuclear localizationpeptide of an active subunit of NF-kB complex. Many genes controlled byNF-kB are known in the art, and others can be readily tested by standardmeans. Examples of such genes include cytokines and interleukins, suchas IL-1, IL-6, granular colony stimulating factor, plasminogen activatorinhibitor and procoagulant tissue factor. Additionally, organisms havinggenes affected by NF-kB can be inhibited by this method, such organismsincluding human immunodeficiency virus (HIV) and cytomegalovirus (CMV).The optimal inhibitory peptide for specific cell types and specificgenes can readily be determined by standard methods given the teachingsherein. Additionally, the optimal inhibitory peptide for a specific celltype subjected to a specific stimulant can readily be determined.

An example is provided herein wherein translocation of the NF-kB complexto the nucleus in endothelial cells stimulated withlipopolysaccharide,(LPS) is inhibited by a complex comprising a signalpeptide linked, to the NLS of subunit p50 of NF-_(κ)B. Presumably, theNLS of subunit p50 interferes with translocation of the complex to thenucleus due to competitive binding. Any cell type subjected to any (orno) stimulus can be readily screened for the optimal inhibitory peptide,i.e., the optimal NLS of a subunit of NF-kB, for that cell type. Forexample, for LEII cells, as demonstrated herein, the NLS of p50 isoptimal.

The subunits of NF-kB complex are known in the art. ⁴³ They include p50,p65 and cellular REL (c-REL). The nuclear localization sequences ofthese subunits are also known. An “active” subunit of NF-kB complex, asused herein, means a subunit which, when it is inhibited, causestranscription factor NF-kB not to function to mediate transcription ofgenes under its control. The nuclear location peptide used in thismethod can be a modification of the known NLS of these subunits are longas it retains the function of inhibiting expression of a gene controlledby NF-kB, as can be readily determined according to the teachings hereinand knowledge in the art.

The invention further provides a method of stimulating the immune systemof a subject comprising administering to the subject a complexcomprising an importation competent signal peptide linked to anantigenic peptide. The complex can be administered to the subject bystandard means known in the art for administering vaccines. The methodcan facilitate uptake of the antigen into cells for subsequent antigenpresentation and the resultant known cascade of the immune system toresult in the stimulation of immunity to the antigen.

Furthermore, if known peptides for blocking auto-reactive T cells arelinked to a signal peptide and administered to a subject, animmuno-suppressive effect can be stimulated in the subject. Such amethod of stimulating immuno-suppression can be used to treat autoimmunediseases such as multiple sclerosis. These blocking peptides can also beadministered by known methods for administering peptides, such asmethods for administering vaccines.

The invention also provides a complex comprising a biologically activemolecule linked to an importation competent signal peptide and to anuclear localization peptide. The linkage can be made as described aboveor otherwise known in the art. Though, as described above, any signalpeptide and any nuclear localization sequence can be utilized, such acomplex is exemplified by the amino acid sequences set forth in SEQ IDNO:3 and SEQ ID NO:4, which contain the K-FGF signal peptide (SEQ IDNO:5) linked to the aFGF nuclear localization peptide (SEQ ID NO:2).

The invention further provides a complex comprising an importationcompetent signal peptide linked to biologically active molecule selectedfrom the group consisting of a nucleic acid, a carbohydrate, a lipid, aglycolipid and a therapeutic agent. This complex can further comprise aliposome. These complexes can be formed as described above. Liposomescan be selected as described above. The complex can be placed in apharmaceutically acceptable carrier.

Treatment of Inflammatory Diseases and Conditions

The methods and peptides of the present invention can be used to treatany inflammatory disease or condition involving pro-inflammatorycytokines.

For example, the invention provides methods for treating or preventingan inflammatory response in a subject. These methods includeadministering to the subject a peptide containing an NF-kB nuclearlocalization sequence such that nuclear import of a stress-responsivetranscription factor is inhibited in a cell of the subject, therebytreating or preventing an inflammatory response in the subject.

As exemplified below, the stress-responsive transcription factor can beNF-kB, AP-1, NFAT, or STAT-1. The NF-kB nuclear localization sequencecan include the amino acid sequence Gln-Arg-Lys-Arg-Gln-Lys or the aminoacid sequence Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro. Also, the peptidecontaining the NF-kB nuclear localization sequence can further include acell membrane-permeable hydrophobic region of a signal peptide.

The peptides of the invention include cyclic peptides that contain acell membrane-permeable hydrophobic region of a signal peptide and anNF-kB nuclear localization sequence. One specific example of such acyclic peptide is cSN50 (SEQ ID NO: 12). For example, the cyclicpeptides of the invention can include a cyclized NF-kB nuclearlocalization sequence having the amino acid sequenceCys-Xaa-Xaa-Gln-Arg-Lys-Arg-Gln-Lys-Xaa-Xaa-Cys(Cys-Xaa-Gln-Arg-Lys-Arg-Gln-Lys-Xaa-Xaa-Cys), wherein Xaa is any aminoacid (for example, any naturally occurring amino acid, or a syntheticvariant thereof). Moreover, one or more amino acids of the cyclicpeptides can be a D-amino acid. The Xaa residues surrounding the NF-kBnuclear localization sequence of the cyclic peptides can all correspondto the sequence found in cSN50 or the sequence found in the naturallyoccurring NF-kB nuclear localization sequence, or only one Xaa, or anycombination of two Xaas, three Xaas, four Xaas, or all five Xaas maydiverge from the sequences found in cSN50 or in the naturally occurringNF-kB nuclear localization sequence. Such cyclic peptide Xaa variantsare made by methods that are well known in the art and tested asdescribed herein, or using any other known assay for measuringinflammatory responses or nuclear import of a stress-responsivetranscription factor. The present invention provides methods foridentifying cyclic peptides for treating or preventing an inflammatoryresponse in a subject, using any of the methods described herein (see,e.g., Example III).

Cyclization can be achieved by other means, as described in Example Vbelow. Cyclic peptides can also be produced by cyclization via residues(e.g., cysteine) at the amino and carboxy termini of the peptide asdescribed in Example V below. The cell membrane-permeable hydrophobicregion of a signal peptide can include a Kaposi Fibroblast Growth Factorsignal peptide hydrophobic region having the amino acid sequenceAla-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Ala-Leu-Leu-Ala-Pro or anintegrin beta-3 signal peptide hydrophobic region having the amino acidsequence Val-Thr-Val-Leu-Ala-Leu-Gly-Ala-Leu-Ala-Gly-Val-Gly-Val-Gly.

The methods and peptides of the invention can be used to treat orprevent inflammatory responses caused by a microbe (or a toxin from amicrobe), e.g., a bacterium (e.g., a Gram-positive or Gram-negativebacterium), a rickettsia, a virus, a fungus, or a protozoan. Forexample, the bacterium can be a Gram-negative bacterium such asEscherichia coli, Salmonella typhimurium, Salmonella typhosa and otherSalmonella species, or Pseudomonas aeruginosa and other Pseudomonasspecies; or the bacterium can be a Gram-positive bacterium, such as aspecies of Staphylococcus, Streptococcus, and Pneumoccocus that causesan inflammatory response, for example, as a result of food poisoning ora noscomial infection). Other examples of microbial infections thatcause inflammatory responses that can be treated or prevented by themethods of the invention include rickettsia, e.g., Rickettsiarickettsiae; viruses, e.g., Ebola virus, Dengue hemorrhagic fever virus,West Nile encephalitis virus, and hepatitis virus A, B, or C; fungi,e.g., Candida albicans, Cryptococcus neoformans, and Histoplasmacapsulatum), and protozoans e.g., Plasmodium falciparum and otherspecies of Plasmodium that cause malaria.

The methods and peptides of the invention can be used to treat orprevent inflammatory reactions triggered by toxins, such as any toxinproduced by a microbe that causes an inflammatory response, for example,but not limited to, lipopolysaccharide, or a superantigen (e.g.,Staphylococcus enterotoxin A or B, streptococcal pyrogenic toxins and Mproteins, or any superantigen produced by a microbe). The methods canalso be used to treat or prevent any inflammatory reaction induced by asuperantigen. Other examples of toxins that trigger inflammatoryreactions that can be treated by the methods of the invention includeplant toxins, e.g., poison ivy or poison oak, nickel, latex,environmental toxins (such as toxic chemicals) or allergens that invokean inflammatory response upon skin contact or inhalation. For example,inhalation of toxins can cause Adult Respiratory Distress Syndrome(which can also result from septic shock and other medical conditions),which can be treated or prevented using the methods of the invention.

Both systemic and localized inflammatory responses can be treated orprevented using the methods and peptides of the invention. For example,the methods can be used to treat or prevent systemic inflammatoryresponse syndrome and/or sepsis syndrome, which, if untreated, can leadto septic shock, which may ultimately result in death. As is well knownin the art and described in the Examples below, bacteremias resultingfrom Gram-negative or Gram-positive infections can cause sepsis syndromeleading to septic shock. One well known example of this process is ToxicShock Syndrome caused by species of Gram-positive bacteria such asStaphylococcus or Staphyloccus.

The methods and peptides of the invention can also be used to treat orprevent inflammatory responses that affect the function of specificorgans or organ systems, for example, but not limited to, the liver,bowel, kidney, joints, skin, pancreas, central nervous system,peripheral nervous system, bladder, or reproductive organs. In somecases, the inflammatory response is caused by an inflammatory disease,for example, an autoimnmune disease. Examples of such autoimmunediseases include, but are not limited to, inflammatory bowel disease,Crohn's disease, glomerulonephritis, multiple sclerosis, lupuserythematosis, rheumatoid arthritis, psoriasis, or juvenile diabetes.The methods and peptides of the invention can also be used to treatchronic or acute inflammatory diseases and conditions of the skin, forexample, psoriasis, eczema, or contact dermatitis.

Moreover, cellular apoptosis induced by inflammatory conditionsinvolving pro-inflammatory cytokines and/or nuclear import ofstress-responsive transcription factors (such as NF-kB, AP-1, NFAT, orSTAT-1) can be inhibited, minimized, or prevented using the methods ofthe invention. For example, as described in the Examples below,apoptosis of liver cells resulting from septic shock can inhibited bythe present methods and peptides. These can be used to inhibit livercell apoptosis caused by other types of acute liver injury resultingfrom inflammation, for example, toxins that poison the liver (oneexample being poisoning by acetominophen) or viruses (such as hepatitisvirus).

The invention provides a method for treating or preventing septic shockin a subject, including delivering to the subject a compound including apeptide including a nuclear localization sequence of NF-kB such thatnuclear import of NF-kB is inhibited, thereby treating or preventingseptic shock in the subject. The peptide used in this and any othermethod of the invention can further include a cell membrane-permeablehydrophobic region of a signal peptide. Furthermore, the present methodmay also inhibit the nuclear import of a member selected from the groupconsisting of AP-1, NFAT and STAT-1, the present method may also inhibitthe nuclear import of each of AP-1, NFAT and STAT1.

The invention also provides a method of importing a biologically activemolecule into the nucleus of a cell in a subject, includingadministering to the subject a complex including the molecule linked toa cell membrane-permeable hydrophobic region of a signal peptide and anuclear localization peptide, thereby importing the molecule into thenucleus of the cell of the subject. For example, the molecule can belinked to a peptide including the amino acid sequence set forth in SEQID NO:12 or SEQ ID NO:13.

In addition, the invention provides a method for treating or preventinga systemic inflammatory response in a subject, wherein the systemicinflammatory response involves import of a stress-responsivetranscription factor into the nucleus of a cell in the subject,including administering to the subject a peptide including a nuclearlocalization sequence of NF-κB, thereby treating or preventing thesystemic inflammatory response in the subject.

The invention further provides a method of inhibiting the import of astress-responsive transcription factor into the nucleus of a cell,including administering to the cell a peptide including a nuclearlocalization sequence of NF-κB, thereby inhibiting the import of thestress-responsive transcription factor into the nucleus of the cell. Ifthe cell is within a subject, the peptide can be administered to thesubject using routine methods. In one particular example of the presentmethod, the cell is a liver cell and administration of the peptide tothe liver cell inhibits apoptosis of the liver cell. The peptide used inthis method can for example, be a cyclic peptide, such as a cyclicpeptide including the amino acid sequence set forth in SEQ ID NO: 12.

The invention further provides a complex including a cellmembrane-permeable hydrophobic region of a signal peptide linked to abiologically active molecule selected from the group consisting of anucleic acid, a carbohydrate, a lipid, a glycolipid and a therapeuticagent. In a particular example, the molecule can be linked to a cyclicpeptide, for example, a cyclic peptide including the amino acid sequenceset forth in SEQ ID NO: 12.

The invention also provides peptides, such as a peptide including theamino acid sequence set forth in SEQ ID NO: 9; a peptide including theamino acid sequence set forth in SEQ ID NO: 12; and a peptide includingthe amino acid sequence set forth in SEQ ID NO:13. These and otherpeptides may be used in the methods of the invention.

Statement Concerning Utility

The present method, which provides an effective method for importingbiologically active molecules into cells, has many uses, both in vivoand ex vivo. Specific utilities using the method are apparent and areexemplified as follows. In vivo, the method can be used to deliver intocells therapeutic molecules, such as peptides and proteins to regulateaberrant functions or to supply deficient cells; DNA for gene therapy(e.g., to provide the CFTR gene in cystic fibrosis patients); RNA forantisense therapy (e.g., to inhibit growth as in inhibiting expressionin cancer cells); and therapeutic agents such as cancer drugs or toxicchemicals (which can be administered in lower dosages with this methodas compared to previous methods not utilizing a signal peptide to moreefficiently enter the cells). Ex vivo, the method allows efficienttransfection of cells without performing cell-damaging procedures.Therefore, this method is useful ex vivo in any method that utilizestransfection, such as transecting reporter genes into cells to screenfor compounds that affect expression of the reporter gene, and fortransfecting bone marrow cells, blood cells, cells of an organ forsubsequent transplantation into a subject or culture cells, with a geneto effect protein expression in the cells.

More specifically, this method can be used for anti-thrombotic therapyby administering functional domains of known cell receptors whichmediate aggregation of platelets, by competitive binding. Additionally,the method can be used for immunosuppression in autoimmune diseases byintroducing immunosuppressive peptides into cells involved in the immuneresponse. Furthermore, growth inhibitors can be administered by thismethod to tumor cells to treat, for example, cancer cells.

This method can also be used to facilitate the absorption ofbiologically active molecules from, e.g., the mouth, stomach orintestinal tract by facilitating movement of the molecules into theconnective tissue beneath the lining of the digestive tract. Also, byallowing one to design signal peptides with modified amino acids, onecan stabilize biologically active peptides by making them more resistantto peptidases and therefore also prolong the action of the peptide.

In addition, methods of treating sepsis are also provided.

As used herein, “a” can mean one or more, depending on the context inwhich it is used.

The invention is more particularly described in the following exampleswhich are intended as illustrative only since numerous modifications andvariations therein will be apparent to those skilled in the art.

EXAMPLE I

The peptides used herein were synthesized by a step-wise solid-phasepeptide synthesis approach²⁴ and purified by high performance liquidchromatography using C₁₈ reverse phase column as described. ²⁵ The exactmolecular weights of the purified peptides were confirmed by massspectrometry analysis.

Amino acid residues 1-16 of the SM peptide were patterned after thepredicted signal peptide sequence of K-FGF^(16,17) (listed separatelyherein as SEQ ID NO:5), residues 17-19 were designed as a spacer, andresidues 20-26 contain an epitope tag recognized by antibody (see SEQ IDNO:1). Amino acid residues 1-19 of the SA peptide are identical to thoseof the SM peptide. However, its carboxyl terminal residues 20-26 aresame as the sequence of the ANL peptide (SEQ ID NO:2), which is derivedfrom the nuclear localization sequence of acidic FGF.¹⁸ The SA peptideis listed as SEQ ID NO:3. The amino acid sequence of the SAα peptide wasthe same as that of the SA peptide except it had a two amino acidresidue extension (Met-Pro) at the carboxyl terminus, which created anepitope (Leu-Met-Pro) for anti-SM peptide antibody. The SAα peptide islisted herein as SEQ ID NO:4.

Membrane-Permeable Signal Sequence Peptide (SM Peptide)

A 26-residue peptide (referred to as SM, listed herein as SEQ ID NO:1)that contained the predicted signal sequence of Kaposi fibroblast growthfactor ¹⁶⁻¹⁷ (K-FGF) was chemically synthesized. An indirectimmunofluorescence assay using antibody against epitope tag-containingSM peptide was employed to follow translocation of the SM peptide to theintracellular compartments of NIH 3T3 cells. A polyclonal anti-SMpeptide antibody against the SM peptide-keyhole limpet haemocyaninconjugate (Pierce) was raised in rabbits and reacted with SM peptide inELISA (titer>1:30,000). The intracellular SM peptide was detected by anindirect imrnmunofluorescence assay using affinity-purified anti-SMpeptide IgG and rhodamine-labeled anti-rabbit antibody (Kirkegaard &Perry). Briefly, confluent NIH 373 cells on the chamber slides (Nunc)were treated with either 0.5 ml SM peptide solution (100 μg ml⁻¹) inDMEM containing 10% FBS or with 0.5 ml DMEM containing 10% FBS only for30 minutes at 37° C. The cells were fixed with 3.5% paraformaldehydesolution in PBS followed by 0.25% Triton X-100 in PBS and then treatedwith 1:20 anti-SM peptide IgG in PBS containing 0.5% bovine serumalbumin (BSA) for 1.5 h. The intracellular SM peptide-antibody complexeswere visualized by subsequent incubation with a rhodamine-labeledanti-rabbit polyclonal antibody for 1 h. In control systems, anti-SMpeptide antibody was preabsorbed with the SM peptide. Intracellularlocalization of the SAα peptide was detected by immunofluorescence assayusing affinity-purified anti-SM peptide IgG as described above for SMpeptide. Following incubation of cells with SM peptide, intracellulardeposits were observed in almost all cells. A ten-step z-positionsectional scanning of the SM peptide-treated cells by the confocal laserscanning microscopy (CLSM) affirmed that these deposits wereintracellular. Immunodetection of the SM peptide was specific becausecells incubated with the peptide-antibody complex showed no evidence ofintracellular peptide. Likewise, cells not exposed to SM peptide orcells exposed to SM peptide followed by the secondary antibody alonewere negative. If cells were fixed with paraformaldehyde before peptidetreatment, the cellular import of SM peptide was prevented.

To determine the rate of SM peptide import across cell membranes, akinetic experiment was carried out with the SM peptide-treated NIH 3T3cells. NIH 3T3 cells were treated at 37° C. with 0.5 ml SM peptide at100 μg mL⁻¹ in DMEM containing 10% FBS for 1, 5, 15, 30, 60 and 120minutes. Intracellular SM peptide deposits were detected by indirectimmunofluorescence assay as described above. The intracellular stainingof intracellular SM peptide was observed during the first 5 minuteinterval and plateaued at about 30 minutes, indicating that the signalsequence-mediated peptide import into cells is rapid. The 30-minute timepoint was therefore selected to determine the optimal peptideconcentrations for detectable import.

To determine the optimal peptide concentration, NIH 3T3 cells weretreated for 30 minutes at 37° C. with 0.5 ml SM peptide solution in DMEMcontaining 10% FBS at the following concentrations: 0, 2, 10, 50, 100,and 150 μg ml⁻¹. Intracellular localization of the SM peptide wasdetected by indirect immunofluorescence assay as described above.Indirect immunofluorescence assay demonstrated detectable peptide in theform of intracellular punctate deposits when the cells were exposed to 2μg/ml (about 800 nM) of peptide. The cellular import wasconcentration-dependent and reached a plateau between 50 μg/ml and 100μg/ml.

Transport of the SM peptide across the cell membrane wastemperature-dependent. No immunofluorescence staining was observed whenthe cells were treated for 30 minutes with 100 μg/ml of the peptide at4° C., whereas, cells treated at either 22° C. or 37° C. showed numerouspunctate deposits. Accordingly, cellular import of the SM peptideresumed when the incubation temperature shifted from 4° C. to 37° C.

Moreover, this signal sequence-mediated import is not limited to NIH 3T3cells. The intracellular localization of SM peptide has been used inbaby hamster kidney-21 cells, human umbilical vein endothelial cells(HUVF-Cs) and rodent endothelial cell line (LE-II), by the aboveindirect immunofluorescence assay, with the same results as with NIH 3T3cells.

Membrane-Permeable Signal Peptide (SKP Peptide)

Intracellular localization of the membrane-permeable peptide was alsoshown by treatment of cells with proteases following peptide import. Forthis experiment, 41-residue peptide (referred to as SEP and listedherein as SEQ ID NO:6) that contained the same hydrophobic sequence asSM peptide followed by the sequence of K-FGF(129-153) was designed andsynthesized. The latter was present in KP peptide not containinghydrophobic sequence that served as the control for themembrane-permeable SKP peptide. Both peptides possess tyrosine residues,therefore they were radiolabeled with ¹²⁵I and examined for theirability to translocate into intact NIH 3T3 cells, as described below.Substantial radioactivity was detected in the ¹²⁵I-SKP peptide-treatedcells but not in ¹²⁵I-KP peptide-treated cells, indicating that theimport of SKP peptide into cells was selectively achieved due to thepresence of hydrophobic, membrane-permeable sequence. The intracellular¹²⁵I-SKP peptide was resistant to the action of proteases. Afiertreatment of cells containing ¹²⁵I-SKP peptide with pronase or trypsin,no significant loss of cell-associated radioactivity was observed,suggesting that the ¹²⁵I-SKP peptide was located in intracellularcompartments (Table 1). The import of membrane-permeable peptide was notdependent on ATP as high energy source because cells depleted of about95% of ATP showed a similar 125I-SKP peptide uptake as compared toATP-positive cells (Table 1).

Both SKP and KP peptides were radiolabeled with ¹²⁵I by the lodogenmethod (Pierce). The specific activities of both peptides were similar(2.5×10⁴ cpm/ng). NIH 3T3 cells were subcultured on a 60-mm dish andincubated at 37° C. for 3 days. The confluent monolayers (1.6×10⁶ cells)on each dish were then washed twice with PBS and treated with 15 ng of¹²⁵I-SKP or ¹²⁵I-KP peptide at 37° C., for the indicated time. The cellswere washed eight times with PBS and twice with 2 M NaCl buffer (pH 7.5)until no radioactivity could be detected in the washings. The washedcells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 1mM phenylmethylsulphony fluoride, 1 mM dithiothreitol, and 1% TritonX-100) and the radioactivity in the cell lysates was counted in aPackard auto-gamma counter. In some experiments, the washed cells werefurther treated with pronase (1 mg/ml) or trypsin (0.05%) solution inDMEM for 5 min at 37° C. The supernatants and cells were separated andtheir radioactivities were counted separately. For ATP depletion assay,cells were incubated with 5 μglml antimycin, 6.5 mM 2-deoxyglucose, and10 mM glucono-δ-lactone in DMEM for 2 h at 37° C. before addition of¹²⁵I-SKP peptide. The levels of ATP in ATP-depleted cells and normalcells were determined by ATP bioluminescent assay kit (Sigma). Nomeasurable ATP was observed in ATP-depleted cells.

TABLE 1 IMPORT OF 12SI-SKP PEPTIDE INTO ATP-DEPLETED NIH 3T3 CELLS ANDEFFECT OF PROTEASES ON CELL-ASSOCIATED ¹²⁵I-SKP PEPTIDE ATP DepletionCounts in cells (cpm/1.6 × −10′ cells) Untreated cells 20,189 ± 2,109 APT-depleted cells 22,266 ± 3,602  Protease Treatment Counts in celllysates Counts in supernatants Untreated cells 21,323 ± 853   2,966 ±838   Pronase 21,791 ± 1,953  1,979 ± 75   Trypsin 23,193 ± 3 10   655 ±70 

For ATP depletion assay, confluent NIH 3T3 cells (1.6×10⁶ cells) in eachdish were treated with or without ATP depleting reagents (antimycin,2deoxyglucose, and glucono-δ-lactone) for 2 h at 37° C. Cells were thentreated with 15 ng of ¹²⁵I-SKP peptide for 30 min at 37° C. Aftercomplete removal of extracellular¹²⁵I-labeled peptides, theradioactivity in the cell lysates was counted. For the assay usingproteases, cells were treated with ¹²⁵ I-SKP peptide and washed asdescribed above. The ¹²⁵I-SKF peptide-associated cells were then treatedwith pronase (1 mg/ml), trypsin (0.05%), or diluent for 5 min at 37° C.The radio-activities in the cell lysates and supernatants were countedseparately. Data in Table 1 represent the mean the mean ±SEM oftriplicate determinations of a single experiment. The experiment wasrepeated three times with similar results.

Membrane-Permeable Signal Peptide with Functional Peptide Cargo (SAPeptide)

Having demonstrated the feasibility of the cellular import of signalsequence-containing peptides, functional cargo in the form of a sequenceresponsible for the nuclear localization of cellular proteins was linkedto a signal peptide. The nuclear localization sequence (NLS) of acidicFGF (aFGF), because it has previously been reported to play an essentialrole in aFGF mitogenic activity, was utilized. ¹⁸ It had previously beenshown that a mutant aFGF with deletion in its NLS regionAsn-Tyr-Lys-Lys-Pro-Lys-Leu (NYKKPKL), listed herein as SEQ ID NO:2,failed to stimulate DNA synthesis and cell proliferation in vitroalthough it could still bind to the FGF receptor and induceintracellular receptor-mediated tyrosine phosphorylation and c-fosexpression. ¹⁸ Additionally, a recent study ¹⁹ of nuclear transport ofaFGF suggested that translocation of aFGF to the nucleus was necessaryfor stimulating DNA synthesis by aFGF in vitro.

A 26-residue hybrid peptide (referred to as SA, listed herein as SEQ IDNO:3) was designed and synthesized. It contains the signal sequence ofK-FGF ^(16,17) at its amino terminal region (residues 1-16 of SEQ IDNO:3) and a “functional cargo” in the form of a nuclear localizationsequence (NLS) of aFGF ¹⁸ at its carboxyl terminal region (residues20-26 of SEQ ID NO:3), separated by a spacer region of Ala-Ala-Ala(residues 17-19 of SEQ ID NO:3). Thus, the SA peptide differs from theSM peptide only in its 7-residue carboxyl terminal “cargo” region. Afunctional assay was performed in which SA peptide was able to induce amitogenic response of NIH 3T3 cells measured by [³H]thymidineincorporation in a manner similar to aFGF bearing, the same NLS. ¹⁸

In this functional assay, confluent 3T3 cells grown initially in DMEMcontaining 10% FBS were transferred to a low serum medium (DMEMcontaining 0.5% FBS) for 2 days. The test peptides, either SA peptide,SAα pep-tide, ANL peptide, or SM peptide, or aFGF, were added to a freshlow serum medium at the indicated concentrations at 37° C. After 16hours, [³H]thymidine was added and 4 hours later, the cells were washedwith PBS, treated with trichloroacetic acid, solubilized with 0.15 MNaOH, and the radioactivity was determined in a liquid scintillationcounter.

As shown in FIG. 1 a, SA peptide stimulated [3H]thymidine incorporation6-fold, while aFGF induced approximately an 8-fold stimulation in thesame assay (FIG. 1 b). Bars represent the mean±S.E.M. of at least threeindependent experiments done in triplicate and calculated asmultiplicity of counts in the tested sample over the control sample. SApeptide within the concentration range used (0 to 46 μM) was notcytotoxic as determined by staining with fluorescein diacetate/ethidiumbromide. ²⁰

Mitogenic Activity of the SA Peptide

To determine whether the mitogenic activity of SA peptide required itsfull length sequence, two control peptides were examined in the sameassay. They are the SM peptide containing signal peptide listed hereinas SEQ ID NO:1) and a 7-residue peptide (referred to as ANL, listedherein as SEQ ID NO:2) representing the NLS of aFGF. Neither controlpeptide showed any significant mitogenic activity when tested withincomparable concentration ranges (FIG. 1 a). These results suggest thatneither the signal sequence alone (SM peptide) nor the nuclearlocalization sequence alone (ANL peptide) was sufficient formitogenesis. SA peptide therefore was effective in mitogenesis becauseit contained both the signal peptide sequence of K-FGF (for import intothe cell) and nuclear localization sequence of aFGF (for mitogenicactivity).

To further confirm the mitogenic activity of the SA peptide, its effecton DNA synthesis was examined. Serum-starved NIH 3T3 cells were treatedwith SA peptide, fixed, and the DNA concentration was determined bystandard flow cytometric analysis. Specifically, confluent NIH 3T3 cells(1.3×10⁶ cells) were serum-starved in DMEM containing 0.5% FBS for 2days. The cells were untreated (control) or treated with SA peptide oraFGF for 20 h, harvested, spun down, and washed with serum-free PBS,three times. The cells were fixed with methanol precooled to −20° C. forDNA analysis carried out by the Flow Cytometry Research Service ofVanderbilt University. The data were the mean ±S.E.M. of sixmeasurements and were analyzed for statistical significance by analysisof variance.

As shown in Table 2, the DNA synthesis in the S-phase of the cell cyclewas significantly increased when the cells were treated with the SApeptide at 100 μg/ml, which coincided with the active concentration inthe thymidine incorporation assay (FIG. 1 a). This result furtherconfirms the role of the NLS region of aFGF in mitogenesis. ¹⁸ Thus,these data also are consistent with a recent demonstration using agenetic approach that schwannoma-derived growth factor requires NLS toexert its mitogenic activity. ²¹

TABLE 2 DNA SYNTHESIS STIMULATED BY SA PEPTIDE AS COMPARED TO aFG1Stimulus Diploid % S Phase Control  7.2 ± 0.7 SA (50 μg ml⁻¹)  6.7 ± 0.9SA (100 μg ml⁻¹) 13.1 ± 0.5 (P < 0.05) aFGF (15 ng ml⁻¹) 27.8 ± 2.3 (P <0.05)

However, compared with aFGF, the SA peptide is less mitogenically potentin both thymidine incorporation and DNA analysis assays (FIG. 1 andTable 2). aFGF binds to the FGF receptors on NIH 3T3 cells and inducesthe tyrosine phosphorylation of a number of intracellular proteins thathave been suggested as the FGF receptor signalling substrates. ^(22,23)In contrast, SA peptide did not stimulate the tyrosine phosphorylationof these intracellular proteins in the same cells even at theconcentrations sufficient to induce DNA synthesis. Taken together, theseresults, make it unlikely that the mitogenic effect of SA peptide wasmediated by FGF receptors.

Immunofluoresence Assay for Modified SA Peptide

The intracellular SA peptide could not be tracked by animmunofluorescence assay because it was not recognized by the availableanti-SM peptide antibody. However, attaching two extra amino acidresidues (Met-Pro) to the carboxyl terminus of the SA peptide produced amodified SA peptide (referred to as SAα, listed herein as SEQ ID NO:4)that contained a 3-amino acid epitope tag, Leu-Met-Pro, recognized byanti-SM peptide antibody in ELISA. Accordingly, intracellular SAαpeptide was observed in a punctate staining pattern in the SAα-treatedNIH 3T3 cells by an indirect immunofluorescence assay using anti-SMpeptide antibody. Like SA peptide, SAα peptide was mitogenic in thethymidine incorporation assay. These results are consistent with therelationship between the SA peptides' import into the intracellularcompartments and their mitogenic activity.

Membrane-Permeable Signal Peptide with Functional Peptide Cargo (SN50)

Having demonstrated the feasibility of the cellular import ofmembrane-permeable SM and SKP peptides, another functional cargo wasattached to the amino-terminal hydrophobic sequence conferring membranepermeable capacity. For this purpose a sequence representing afunctional domain of the nuclear factor kB (NF-kB) responsible for anuclear localization signal was selected. Import of such a peptide intothe cell would be measured by inhibition of nuclear translocation ofNF-kB complex in stimulated cells. The NF-kB is a pleiotropic activator^(39,40) that plays a critical role in the regulation of a number ofcellular and viral genes, including the enhancer of humanimmunodeficiency virus (HIV). The inactive cytosolic form of NF-kB is aheterotrimer including p⁵⁰, p⁶⁵ and an inhibitory protein IkB.^(41,42)Upon activation of cells with stimuli such as lipopolysaccharide (LPS)or cytokines, ^(43,44,45)IkB dissociates from the complex. Thisdissociation allows the translocation of heterodimer of p50 and p65subunits to the nucleus. Both p50 and P65 subunits contain NLS,suggesting that the NLS sequence may be important for nuclear uptake ofNF-kB.

To determine the functional significance of the NLS of p50 and p65subunits, two peptides were designed and synthesized containing thesequence motifs. The first peptide (referred to as SN50, listed hereinas SEQ ID NO:9) contained the signal sequence of K-FGF^(16,17) at itsamino-terminal region (residues 1-16) and a “functional cargo” in theform of NLS of NF-kB p50 subunit at its carboxy-terminal region(residues¹⁷⁻²⁶). The second peptide is also a 26-residue peptide(referred to as SN65, listed herein as SEQ ID NO:8) that contains thesame hydrophobic sequence and the NLS of p65 subunit. Both peptides weretested for their inhibitory effects on the nuclear translocation of theNF-_(κ)B complex in LE-II cells. Inducible _(κ)B binding activity wasdetectable by electrophoretic mobility shift assay in nuclear extractsfrom cells treated with LPS for 1 h. ⁴³ However, this LPS-induced _(κ)Bbinding activity in nuclear fraction was reduced substantially in theSN50 peptide-treated cells. The inhibition by SN50 peptide wasconcentration-dependent, reading an 88% inhibition at 50 μg/ml. Incontrast, no inhibition was observed in SN65 peptide-treated LEII cells.To exclude the possibility that the inhibition was caused by theinterference of SN50 peptide in the binding of oligonucleotide probe tothe NF-kB complex, SN50 peptide was incubated in vitro with nuclearextracts and radiolabeled probe. This maneuver was without anymeasurable effect on LPS-induced kB binding activity, suggesting thatinhibition by SN50 peptide resulted from its action at the stage inwhich the active NF-kB complex moves from cytosol to nucleus. Todetermine whether the inhibition by SN50 peptide required a hydrophobic,membrane-permeable sequence, two control peptides (SM and N50 peptides,listed herein as SEQ ID NOS: 1 and 10, respectively) were also tested inthe same mobility shift assay. N50 peptide contained the NLS without thehydrophobic sequence, whereas SM peptide contained a hydrophobicsequence without the NLS. Neither of these two peptides showed anysignificant effect on LPS-induced intracellular translocation of theNF-kB complex from the cytosol to the nucleus. These results suggestthat neither the hydrophobic sequence alone (SM peptide) nor the nuclearlocalization sequence alone (N50 peptide) was sufficient for causing afunctional inhibition of the NF-kB. Therefore, the observed inhibitoryeffect of SN50 must be attributed to its intracellular import whichallowed the interaction of its intrinsic NLS with the nuclear membranes.

SN50 peptide contained the same epitope tag as SM peptide and thus couldbe recognized by the anti-SM peptide antibody in ELISA. This alloweddirect affirmation by an indirect immunofluorescence assay that SN50peptide was imported into LE-II cells to exert its functional role.Results showed that the intracellular SN50 peptide was distributed in amore nuclear staining pattern as compared to the intracellular SMpeptide.

EXAMPLE II

The efficacy of the intracellular inhibition of nuclear import of NF-κBand other stress-responsive transactivators in abrogating in vivochanges resulting in lethal septic shock is based on the use ofnoninvasive intracellular delivery of the SN50 peptide containing a cellmembrane-translocating sequence and NLS domain. ⁵⁰⁻⁵² Two cell-permeablepeptides, SM and SN50 were synthesized and purified as previouslydescribed, ^(51,52) and first tested in vitro in two types of cellsknown to be a target for LPS: murine macrophage J774 and endothelialLEII cell lines. (SM and SN50 peptides, listed herein as SEQ ID NOS: 1and 9). The nuclear import of transcription factor NF-κB induced byseptic shock inducer, LPS (10 ng/ml) in murine J774 macrophages wasblocked by SN50 peptide but not by SM peptide (both at 31 μM). Whereasboth peptides are cell permeable, the SM peptide functional domaincontains “loss of function” mutated NLS sequence. ^(51,52) Similarpattern of results was obtained when J774 cells were stimulated withproinflammatory mediator of septic shock, TNFα (20 U/ml). The SN50peptide also inhibited inducible nuclear import of NF-κB in murineendothelial cells LEII stimulated with LPS (10 ng/ml) and TNFα (100U/ml). Thus, the SN50 peptide but not the SM peptide causedintracellular inhibition of inducible nuclear import of NF-κB in murinemacrophages and endothelial cells stimulated by LPS and TNFα.

The efficacy of the SN50 peptide-directed inhibition of NF-κB and otherstress-responsive transactivators in attenuating or preventing in vivoseptic shock was demonstrated by injecting C57BL/6 miceintraperitoneally with D-galactosamine (20 mg) followed by LPS fromE.coli serotype 0127:B8 (1 μg). Mice treated with D-galactosamine aresensitive to low doses of LPS. ⁶⁰ As shown in FIG. 2A all but one mouseshowed symptoms of acute illness within 4 hours and died within 6 hoursfollowing injection of LPS. In contrast, as shown in FIG. 2C, micetreated with the SN50 peptide (5 injections up to 3½ h after LPS) showedno symptoms of shock and survived the first 24 h. By 48 h, 50% micesurvived and by 72 h 20% survival was observed. The protective in vivoeffect is dependent on functional NLS domain of the SN50 peptide,because it was abrogated when SM peptide with mutated NLS domain wasused. All SM peptide-treated mice (5 injections up to 3½ h after LPS)showed symptoms of acute illness and died within 5 h (FIG. 2B). The invivo protective effect of SN50 peptide was time-andconcentration-dependent. Administration of SN50 peptide extended to 6and 12 h following LPS (7 intraperitoneal injections) resulted in 64%survival at 72 h (FIG. 2D). The differences in survival arestatistically significant with P<0.001 based on the log rank test. ⁶⁰

As this model of septic shock is characterized by fulminant liverinjury, histopathologic analysis focused on the liver. Sections obtainedfrom mice receiving lethal combination of D-galactosamine and LPSrevealed diffuse hepatocellular injury with hallmarks of apoptosis(fragmented nuclei), engorgement of blood vessels filled with plateletthrombi, and extravasation of red blood cells. Identical pattern ofmassive apoptosis of hepatocytes accompanied by hemorrhagic livernecrosis was observed in mice treated with SM peptide. In contrast,tissue sections from the liver of SN50 peptide-treated mice thatsurvived septic shock for 72 h, displayed almost normal liverarchitecture without any overt signs of apoptosis of hepatocytes andhemorrhagic necrosis. The simplest implication of these results is thatintraperitoneal administration of the SN50 peptide provides significantcytoprotection of the liver, a primary target organ in murine model ofD-galactosamine/LPS-induced septic shock. ^(57,60)

These evidence that the SN50 peptide administered in vivo attenuatesand/or prevents lethal septic shock induced by LPS. The microbialinducer of septic shock, LPS, acts on monocytes, macrophages,granulocytes, and endothelial cells that express multiple genes encodingproinflammatory cytokines (TNFα, IL-1, 6, 8, and 12), signal transducers(iNOS and COX2), cell adhesion molecules (E-selectin, VCAM, ICAM) andprocoagulant molecules (Tissue Factor, Plasminogen Activator Inhibitor).^(48,49,67,68) Persistent expression of stress-responsive gene productschanges the quiescent phenotype of blood cells and vascular endotheliuminto an “activated” phenotype and contributes to irreversible vasculardysfunction and leads to the ultimately fatal outcome. ^(47,49) NF-κB isa primary intracellular mediator of signaling to the nucleus induced byLPS in humans and mice wherein persistent nuclear translocation of NF-κBcorrelated with lethal outcome. ⁴⁹ Unlike extracellular LPS inhibitorsor cytokine receptor antagonists, the SN50 peptide blocks the pre-finalobligatory step in intracellular signaling to the nucleus by NF-κB andother stress-responsive transactivators, regardless of the initiatingstimulus. ^(50,52)

The in vivo mechanism of protective action of the SN50 peptide can bededuced from these studies. First, the SN50 peptide is likely toattenuate NF-κB-dependent expression of cytokines and otherstress-responsive gene products contributing to cellular and molecularpathogenesis of septic shock. The inhibitory effect of the SN50 peptideon expression of genes regulated by NF-κB and other transactivators wasdemonstrated. ⁵² Second, it is unlikely that the primary cellular siteof action of the SN50 peptide are LPS-stimulated peritoneal macrophagesbecause they do not appear to be the major cells responsible for theoverall host response during endotoxic shock. ⁶⁹ Rather, we have proventhat the SN50 peptide exerts its protective effect systemically beingabsorbed from peritoneal cavity and crossing plasma membrane of vascularendothelial cells as well as of blood monocytes and tissue macrophages.Therein, it reaches its intracellular target, importin-α and -βheterodimer (also called karyopherin-α and -β) shuttling NF-κB, AP-1,NFAT and STAT1 to the nucleus. ⁵² Significant cytoprotective effect inthe liver and overall gain in survival following repeatedintraperitoneal administration of the SN50 peptide indicate its systemicin vivo effect. Third, the observed in vivo cytoprotective effect of theSN50 peptide in the liver is remarkable in the context of the reportedJanus-like role of NF-κB in preventing and inducing apoptosis. ⁶³Time-dependent anti-apoptotic effect of the SN50 peptide in the liverindicates that LPS and proinflammatory cytokine mediators, eg. TNFα andinterferon γ, cannot induce apoptosis when nuclear import of NF-κB,AP-1, NFAT and STAT-1 is blocked by the SN50 peptide as previouslydemonstrated. ⁵² Other studies using genetically-engineered mice haveprovided significant insight in molecular mechanism of LPS-inducedseptic shock by pinpointing LPS receptors, cytokine receptors, andintracellular caspases as essential molecular mediators of septic shock^(54-59,70-73).

Methods

Animals and Treatment

C57BL/6 mice were obtained from the Jackson Laboratory. 10-12 wk oldfemale mice were injected intraperitoneally with 0.2 ml suspension ofLPS from E. coli 0127:B8 (prepared by phenol extraction and gelfiltration chromatography, Sigma Chemical Co.). D-galactosamine (20 mgin 0.2 ml; Sigma Chemical Co.) was injected intraperitoneally 30 minutesbefore LPS. Cell permeable SN50 and SM peptides (2 mg each in 0.2 ml) or0.9% saline (diluent) were injected 30 min before and 30, 90, 150, 210min after LPS. Additional intraperitoneal injections of the SN50 peptideto the survivors were administered 6 and 12 h after LPS. All injectedagents were sterile and prepared in pyrogen-free saline for injections.Animals were observed at 2 h intervals during the first 12 h, at 4 hintervals during the subsequent 12 h and twice daily thereafter. Tissuesections from the liver, spleen, lungs, and kidneys were examined. Micereceiving either D-galactosamine (20 mg) alone (n=5) or LPS (1 μg) alone(n=5) survived and were unaffected on the basis of tissue sections fromthe principal organs. Animals were handled and experimental procedureswere conducted in accordance with the American Association ofAccreditation of Laboratory Animal Care guidelines and approved by theInstitutional Animal Care Committee.

Cell-Permeable Peptides

The SN50 and SM peptides were synthesized, filter-sterilized, andanalyzed as described. ^(51,52)

Cell Cultures

Murine macrophage RAW 264.7 cell line obtained from the American TypeCulture Collection (Rockville, Md.) and murine endothelial (LEII) cellsobtained from Dr. T. Maciag (Maine Medical Center, Portland, Me.) werecultured in Dulbecco Minimal Essential Medium supplemented with 10%heat-inactivated fetal bovine serum containing no detectable LPS (<0.006ng/ml as determined by the manufacturer, Atlanta Biological, Norcross,Ga.), 2 mM L-glutamine and antibiotics as recommended by ATCC. Whereindicated, 5 ml of RAW 264.7 or LEII cells (10⁶/ml) were stimulated withLPS from Escherichia coli 0127:B8 (Difco) or TNFα with the potency of32,000 U per μg (Mallinckrodt) at concentrations and tissue indicated inthe text.

Electrophoretic Mobility Gel Shift Assay (EMSA)

To measure nuclear import of NF-κB in RAW 264.7 and LEII cells, EMSA wasperformed as described using a radiolabeled κB probe. ^(51,52)

Statistical analysis The log rank test was used to determine P valuesfor mouse survival data. ⁶¹

EXAMPLE III

In this experiment, the effectiveness of this class of cell-permeablepeptides in vitro with murine macrophages and endothelial cells wasmeasured using SN50, carrying a nuclear localization sequence (NLS)derived from NF-κB1 (p50), SM, were synthesized and purified aspreviously described, ^(51,52) and a third cyclic peptide (cSN50) wasdesigned by inserting two cysteines flanking NLS motif to form anintrachain disulfide bond. (cSN50 peptide, listed herein as SEQ ID NO:12) All three peptides are cell-permeable, but the SM peptide functionaldomain contains a mutated NLS that is not recognized by importin α (alsocalled karyopherin α or Rch 1). ⁵² In murine J774 macrophages, nuclearimport of NF-κB in response to LPS (10 ng/ml) was blocked by SN50 butnot SM (both at 100 μM). Similar results were obtained when J774 cellswere stimulated with TNFα (20 U/ml), a proinflammatory mediator ofseptic shock. The SN50 peptide also inhibited inducible nuclear importof NF-κB in murine endothelial LEII cells stimulated with LPS (10 ng/ml)and TNFα (20 U/ml). The third peptide, cSN50 containing a cyclized NLSdomain, was inhibitory at a concentration of 10 to 30 μM. Based onquantitative phosphoimager analysis of these results, the potency ofcSN50 is 3-10 times greater than the prototypical SN50 peptide. Thus,cell-permeable peptides inhibited nuclear import of SRTFs inendotoxin-responsive cells in vitro.

cSN50 was tested in vivo using a murine model for lethal endotoxicshock. ⁶⁰ In this model, LPS from E. coli serotype 0127:B8 (LD₁₀₀=800μg) was injected intraperitoneally into C57BL/6 mice. As shown in FIG.3A, all animals died within 72 hrs following LPS injection. In contrast,mice treated with cSN50 (0.7 mg given in 7 injections 30 minutes beforeto 12 hrs after LPS challenge) were protected from septic shock asevidenced by the lack of its typical signs (piloerection, lethargy,diarrhea, hemorrhagic conjunctivitis, hemorrhagic skin necrosis, andparalysis). During subsequent 24 hrs 1 animal died and after 48 hrs 4died, yielding a 50% survival rate (FIG. 3B). The protective effect ofcSN50 was improved when the dose was increased to 1.5 mg per injection(FIG. 3C). All but one animal survived for 72 hrs (90% survival).Survivors observed for the subsequent 10 days showed no apparent signsof disease. This in vivo protective effect was lost if the functionalNLS motif was mutated as in the SM peptide. All SM peptide-treated micedied within 72 hrs (FIG. 3D). Based on the log rank test, the differencein survival rate between cSN50 peptide-treated groups and controls wasstatistically significant (p<0.001). Histologic examination of excisedorgans (lungs, liver, spleen, and kidneys) showed minimal changes incSN50 peptide-treated survivors whereas untreated mice dying fromendotoxin showed particularly prominent distention and engorgement ofpulmonary vessels.

Prior studies have shown that following injection of LPS into humans andanimals there is an early burst of proinflammatory cytokine mediators ofseptic shock such as TNFα, IL-1, and INF-γ.(75) To determine whether thecSN50 peptide reduces lethality when administered after exposure toendotoxin, the first peptide dose was given 30 min after endotoxin. Thesurvival rate was 60%, indicating that the cSN50 peptide may exert itsprotective effect, if given shortly after exposure to endotoxin. Takentogether, cSN50 peptide protected mice from endotoxin-induced lethalshock in a time and concentration-dependent manner. Increasing the doseof injected peptide and/or number of injections improved survival rate.The requirement for repeated administration of cell-permeable peptideindicates that its protective effect is transient; reducing the numberof injections lowers the survival rate consistent with a relativelyshort intracellular half-time (˜45 min) of SN50.⁵⁰ Thus, the rapidlyreversible effect of cell-permeable peptide accounts for its short-termeffectiveness and safety. No lethality or tissue injury was observed inanimals receiving peptide alone.

The efficacy of the cell-permeable peptide described in this studylikely reflects in vivo inhibition of signaling to the nucleus mediatedby SRTFs. ^(51,52) In the absence of nuclear import inhibitor, SRTFspotently stimulate transcription of the genes encoding pro-inflammatorymediators of lethal shock.^(48,68,77) In turn, the persistent expressionof these genes in monocytes, macrophages, granulocytes, and endothelialcells is associated with profound vascular dysfunction and death.^(48,49,57,68,77) The inhibitory effect of the SN50 peptide on theexpression of genes regulated by NF-κB and other SRTF has beendemonstrated. ⁵² The finding that the cell-permeable peptides carryingNLS inhibit nuclear import in vitro shows that they can preventLPS-induced activation of these inflammatory stress-responsive genes invivo. Consistent with this, mutations that inactivate NLS function yielda cell-permeable peptide (SM) that fails to affect the acute systemicinflammatory response to endotoxin.

These experiments provide a conceptually novel approach to treatment ofendotoxic shock. In contrast to extracellular inhibitors of LPS orcytokine receptor antagonists, ⁷⁵ nuclear import inhibitors of SRTFs aretargeted intracellularly. ⁵⁰ The multiple proinflammatory agonists, egLPS and cytokines, upon binding to their cognate receptors, initiate acascade of signaling steps converging at the common step of nuclearimport of SRTFs. ^(48,54-58,68,77) Reversible inhibitors of nuclearimport exemplified by cSN50 constitute a new class of anti-inflammatoryagents capable of suppressing a systemic inflammatory response.Consistent with this approach, the SN50 peptide was effective inblocking lethal shock induced by superantigen, staphylococcalenterotoxin B, interacting with murine T lymphocytes. ⁷⁶ In conclusion,our results with the NLS peptide functioning as nuclear import inhibitorprovide a new, effective, and convenient in vivo targeting strategy toreduce morbidity and mortality in the systemic inflammatory responsesyndrome exemplified by endotoxic shock.

Methods

Animals and Treatment.

C57BL/6 mice were obtained from the Jackson Laboratory. 8-12 wk oldfemale mice (20 g weight) were injected intraperitoneally with 0.2 mlsuspension of LPS (800 μg) from E. coli 0127:B8 (Difco, Detroit, Mich.).Cell-permeable cSN50 and SM peptides or 0.8% saline (diluent) wereinjected 30 min before and 30, 90, 150, 210 min, 6 hrs and 12 hrs afterLPS. In some experiments cSN50 peptide was not injected before LPS. Allinjected agents were sterile and prepared in pyrogen-free saline.Animals were observed at 2 h intervals during the first 8 h, at 4 hintervals during the subsequent 16 h, and twice daily thereafter.Autopsies were performed shortly after death or after sacrifice at 72hours. Surviving animals were observed for 10 days. Animals were handledand experimental procedures were conducted in accordance with theAmerican Association of Accreditation of Laboratory Animal Careguidelines and approved by the Institutional Animal Care Committee.

Cell-permeable Peptides

The SN50 and SM peptides were synthesized, filter-sterilized, andanalyzed as described.^(11,12,51,52) The cSN50 peptide was synthesizedand analyzed in a similar manner.

Cell Cultures

Murine macrophage J774 cell line was obtained from the American TypeCulture Collection (Rockville, Md.) and murine endothelial (LEII) cellswere kindly provided by Dr. T. Maciag (Maine Medical Center, Portland,Me.). Both cell lines were cultured in Dulbecco Minimal Essential Mediumsupplemented with 10% heat-inactivated fetal bovine serum containing nodetectable LPS (<0.006 ng/ml as determined by the manufacturer, AtlantaBiological, Norcross, Ga.), 2 mM L-glutamine and antibiotics. Whereindicated, 80% confluent monolayers of J774 or 100% confluent LEII cells(100 mm plates with 10 ml fresh medium) were stimulated with LPS fromEscherichia coli 0127:B8 (Difco) or with TNFα (32,000 U per μg;Mallinckrodt) at concentrations and time indicated in the text. Nuclearimport of NF-κB in J774 and LEII cells was measured by Electrophoreticmobility gel shift assay (EMSA) using a radiolabeled κB probe. ^(6,7)

Electrophoretic Mobility Gel Shift Assay (EMSA)

Measurement of the nuclear import of NF-κB was performed as describedusing a radiolabeled κB probe. ^(51,52)

Histologic Analysis

Formalin-fixed, paraffin-embedded sections of the liver, spleen, lungs,and kidneys were stained with hematoxylin and eosin to assess overallhistology.

Statistical Analysis

The log rank test was used to determine P values for mouse survivaldata.⁶¹

At the molecular level, systemic inflammatory response syndromes such asendotoxic shock are mediated via nuclear signaling of NF-κB and otherstress-responsive transcription factors (SRTFs), which regulate theexpression of septic shock mediators.^(48,49) These findings demonstratethe in vivo utility of cell-permeating peptide inhibitor of NF-κB in thetherapeutic control of an acute systemic inflammatory response at thelevel of nuclear signaling. Inhibition of nuclear import of SRTFs with acyclic cell-permeable peptide demonstrates a new approach to the controlof systemic inflammatory response syndromes such as endotoxic shock.

Septic shock triggered by endotoxic lipopolysaccharide (LPS) is anextreme form of the systemic inflammatory response syndrome that ischaracterized by collapse of the circulatory system, disseminatedintravascular coagulation, and multiple organ failure resulting in highmorbidity and mortality.^(46,47) Treatment of septic shock is oftenineffectual, as diverse mediators lead to fatal outcome.^(53,66) Thesemediators are expressed because SRTFs relay signals to the nuclei inendotoxin-responsive cells (monocytes, macrophages, endothelialcells).^(48,68,77)

There is abundant evidence that SRTFs, reaching the nuclei from thecytoplasm, activate genes encoding proinflammatory cytokines such as,tumor necrosis factor α (TNFα), interleukins 1,6,8,12,18, cell adhesionmolecules, ICAM-1, E selectin and VCAM, as well as the procoagulantmolecules, tissue factor and plasminogen activator inhibitor.^(48,68,77)The SRTFs, mediating responses to inflammatory and immune stress areNF-κB, AP-1, NFAT and STAT-1.^(48,49,57,67,68,77) For example, NF-κB andNFAT regulate genes encoding primary cytokine mediators of septic shock,TNFα and interferon γ (INF-γ).⁷⁴ The gene encoding tissue factor, aprimary procoagulant mediator of disseminated intravascular coagulation,is regulated by NF-κB and AP1.⁶⁸

In humans and mice, persistent nuclear translocation of NF-κB inmononuclear phagocytic cells correlated with lethal outcome of septicshock.⁴⁹ Nuclear import of these transactivators can be blocked bynon-invasive intracellular delivery of SN50 peptide bearing a membranetranslocation motif and a functional domain comprised of the nuclearlocalization sequence (NLS) derived from NF-κB and recognized byheterodimer of importin a and importin β (also called karyopherin-α and-β). ⁵⁰⁻⁵² These in vivo findings present a novel approach to thetherapeutic control of lethal septic shock involving the delivery ofcell-permeable peptides that affect nuclear targeting of NF-κB and otherstress-responsive transactivators.

EXAMPLE IV Inhibition of Superantigen-Induced Toxic Shock and AcuteLiver Injury by a cSN50 Peptide

Bacterial superantigens (SAgs) are exotoxins produced by Gram-positivebacteria such as staphylococci and β-hemolytic streptococci. Thesetoxins, which include staphylococcal enterotoxins, Toxic Shock SyndromeToxin-1, and streptococcal pyrogenic exotoxins, induce Toxic ShockSyndrome in humans and in animals. SAgs released as a consequenceinfection by Gram-positive bacteria can stimulate a relatively largepercentage (about 10-50%) of all T cells in the body of the infectedindividual, and activation of these T cells leads to systemic cytokineproduction (TNF-α, IL-2, IFN-γ). Such SAg-induced activation of T cellsrequires the presence of antigen presenting cells (APC) expressing classII MHC molecules. The resultant systemic inflammatory response ischaracterized by desquamation, vascular injury, hypotension, anddisseminated intravascular coagulation (DIC). Together, these effectsproduce lethal toxic shock; mortality due to staphylococcal-inducedToxic Shock Syndrome is about 5%, and mortality due tostreptococcal-induced Toxic Shock Syndrome is about 30-80%.

At the molecular level, T cell signaling to the nucleus via the T cellreceptor/CD3 complex induces pro-inflammatory cytokine expression. Thissignaling is mediated by NF-κB and other stress responsive transcriptionfactors (SRTF), including AP1 and NF-AT. The NF-κB p50/p65 heterodimeris complexed with the inhibitory protein, IκB. Cellular activationresults in phosphorylation and then degradation of IκB, therebyreleasing the NF-κB p50/p65 heterodimer for import to the nucleus. AP-1proteins, c-Fos and c-Jun, are present at low levels in resting T cells.De novo protein synthesis is followed by nuclear import of c-Fos andc-Jun. NF-AT is a phosphoprotein in the cytoplasm of resting T cells.Activation of T cells induces its dephosphorylation and nuclear import.NFAT binds to DNA alone or in complex with c-Fos and c-Jun. Thetranscription factors enter the nucleus, bind to the promoters ofpro-inflammatory genes and induce expression of cytokines, such asTNF-α, IL-2, and INF-γ. The newly expressed TNF-α binds to its receptorand induces another cycle of activation. Thus, inhibition of nuclearimport of NF-κB and other stress-responsive transcription factors by acell-permeable peptide analog of the NF-κB nuclear localization sequence(NLS) can suppress expression of genes that encode mediators of toxicshock.

To study the effect of the cSN50 peptide on NF-κB nuclear import innatural killer T (NK-T) cells, we incubated dendritic cells (DC) withstaphylococcal enterotoxin B (SEB) for 60 min at 37° C. and separatelyincubated NK-T cells with cSN50 peptide for 30 min at 37° C., then mixed95% of NK-T cells with 5% of DC and incubated the cell mixture for 2 hrsat 37° C. After incubation, nuclear extracts were prepared and analyzedby electrophoretic mobility shift assay (EMSA) using a probe containinga binding site for NF-κB. SEB-stimulated NK-T cells displayed highlevels of NF-κB translocation to the nucleus, as evidenced by a strongEMSA signal, compared with unstimulated cells, as evidenced by very lowlevels of NF-κB binding to the probe (FIG. 4; compare first and secondlanes). Increasing concentrations of the cSN50 peptide (from 3 to 30 μM)resulted in increasing inhibition of SEB-induced NF-κB nuclear import(FIG. 4; see third through fifth lanes). As a control, included in allEMSA reactions was a probe containing a binding site for NF-Y, aconstitutively expressed nuclear protein. This control shows equalloading of the lanes shown in FIG. 4. These results indicate that thecSN50 peptide inhibited NF-κB nuclear import in NK-T cells.

We next established a murine model of SAg-induced toxic shock, whichinvolves administration of staphylococcal enterotoxin B (SEB) (300 ug)to C57Bl/6 mice (wild type), together with D-galactosamine (20 mg) as asensitizing agent. To study the development of toxic shock, we monitoredthe expression of the pro-inflammatory cytokines TNF-α and IFNγ, mousesurvival rate, and histology. To study the in vivo effect of cSN50peptide on toxic shock development, we used wild type mice, given 7injections IP with 0.7 mg cSN50 peptide. The schedule for cSN50 peptidetreatment was 30 min before SEB and after SEB challenge at 30, 90, 150,210 mins, 6 hr, and 12 h (FIG. 5). As a negative control, we injectedadditional mice with the inactive cell-permeable peptide SM (describedabove), which has a mutated NLS sequence. The peptide-injected mice wereobserved for signs of illness and/or survival for 72 h.

The SEB-challenged, cSN50 peptide-treated mice displayed a survival rateof 80% (FIG. 5). By contrast, the SEB-challenged, SM peptide-treatedmice displayed a survival rate of only 10%, similar to that ofSEB-challenged mice not treated with peptide. These results show thatthe cSN50 peptide protects mice from SEB-induced toxic shock.

The most striking feature of this model of toxic shock is acute liverinjury with apoptosis and hemorrhagic necrosis (FIG. 6A-6D). The leftpanels (FIGS. 6A and 6D) show liver sections from untreated control micechallenged with SEB, stained with hematoxylin and eosin and Apop Tag(Intergen, Purchase, N.Y.), respectively, reveal extensive hemorrhageand apoptosis. In contrast, liver sections from cSN50 peptide-treatedmice challenged with SEB, shown on the right (FIGS. 6B and 6D), displayno detectable signs of liver injury or apoptosis, indicating that thecSN50 peptide provided effective protection from the inflammatoryeffects induced by a SAg from a Gram-positive bacterium.

EXAMPLE V Methods for Producing Cyclized Peptides

Selected peptide sequences of the type X₁-X₂, wherein X₁ contains amembrane-permeable motif, and X₂ contains a nuclear localizationsequence (NLS), can be flanked by two cysteine residues either as:X₁-CysX₂Cys or CysX₁-X₂Cys. Such positioning of the cysteine residuesallows efficient disulfide bond formation and cyclization of either thefunctional segment (X₂) or the entire bipartite peptide (X₁-X₂).Alternatively, lactam or lactone cyclization can be performed bysubstituting cysteine at the N-terminus of X₂ with serine (lactone) orwith diaminopropioric acid (lactane). As shown in Example III above,constraint of the biologically active segment (containing, e.g., anuclear localization sequence, enhances the activity of the peptide.Moreover, the degradation rate of cyclic peptides is distinctly slowerthan that of linear peptides, because the breakdown of a peptide chainproceeds most readily from either the amino or carboxy terminus.

Another approach to cyclization of NLS-containing peptides is based on asolid-phase intramolecular chemical ligation strategy to synthesizecyclic thioester peptides via thiolactone ring formation. A fullyunprotected peptide is immobilized on a solid support through a reactivethiol ester bond. Preloadedt-butoxocarbonyl-aminoacyl-3-mercapto-propionamide-polyetheleneglycol-poly-(N,N-dimethylacrylamide) (Boc-AA-[COS]-PEGA) resin is usedfor synthesis (Camarero, J. A., et al., J. Pept. Res. 51:303-316, 1998and Schnoülzer, M. et al., Int. J. Pept. Protein Res. 40:180-193, 1997)Peptide-[COS]-PEGA resin is treated with HF for 1 hr at 4° C. to obtainfully unprotected peptide. Such an unprotected peptide-[COS]-PEGA resin,containing an ester linkage which anchors peptide to resin, is stable inanhydrous HF and can be then selectively cyclized and simultaneouslycleaned from the resin by its swelling in aqueous buffer (0.1 M sodiumphosphate, pH 7.0 and acetonitrile in the ratio 80:20). After washingthe resin with 0.1% TFA in water, the cyclized peptide is purified byreverse-phase HPLC.

Still another strategy for peptide cyclization is based on the generalmethod of “backbone cyclization” (Gilon, C. et al. Biopolymers31:745-750, 1991) in which the connection of the N^(α) or C^(α) atoms inthe peptide backbone to each other or the carboxyl and amino terminiprovide a constrained conformation of biologically active peptidesegment.

In general, peptides are synthesized using either N-tert-butoxycarbonyl(t-Boc) or the N-9 flourenylmethoxycarbonyl (Fmoc) strategies. Cysteineresidues can be protected with acetaminidomethyl (Acm). Boc deprotectionis performed with 50% trifluoroacetic acid (TFA) in dichloromethane(DCM). Fmoc deprotection is performed with 20% piperidine in DMF for 30min and repeated twice each time. Peptides from Fmoc synthesis arecleaved from the resin by TFA/thioanisole/triisopropylsilane/methanol(90:5:2.5:2.5; vol/vol/vol/vol) at 20° C. for 4 hrs. Peptides from Bocsynthesis are cleaved by anhydrous fluorhydric acid (HF)/anisole (9:1vol/vol) at 4EC for 1-2 hr and the crude peptides are precipitated withcold ethyl ether, dissolved in 60% acetonitrile in H2O and lyophilized.Peptides are dialyzed against water using a Spectra/Por CE dialysismembrane (molecular weight cut off: 500) and chromatographed on highpressure liquid chromatography. A reverse phase (HPLC) column (VydacC-18; 0.045% TFA in water/acetonitile gradient). The bis (Acm)-Cysprotected peptides can be cyclized, e.g., in 8:1 acetic acid/water withiodine as described (Kamber, B. et al., Helv. Chem. Acta 63:899-915,1980). The completeness of cyclization can be assessed by electrospraymass spectrometry (loss of Acm groups) and a negative Ellman's test. Thepurified peptides are analyzed by analytical HPLC, matrix-assisted laserdesorption ionization mass spectroscopy (MALDI-MS), and amino acidanalysis.

EXAMPLE VI Inhibition of NF-κB Nuclear Import by D-aminoAcid-Substituted MPS-NF-κB Peptides

The fundamental mechanism underlying the transport of functionalpeptides across plasma membrane barrier remained unexplained. While notwishing to be bound by theory, we hypothesized that the intracellulardelivery of our cell-permeable peptides across the plasma membranes ofmultiple cell types involves translocation through the membranephospholipid bilayer, rather than receptor- or transporter-specificrecognition and uptake. To test this hypothesis, we synthesized,purified, and tested the enantio-inverso (all D-amino acids) analog of ahydrophobic motif signal sequence, which we had previously designed as amembrane-permeable sequence (MPS).

MPS based on the hydrophobic region of the signal sequence of KaposiFibroblast Growth Factor (KFGF), as described above, was synthesizedwith all L- or all D-amino acids to establish whether the import isdependent on chirally-specific receptor or membrane transport. All L- orits “mirror image” all D-MPS was coupled to functional domain (“cargo”)containing nuclear localization sequence (NLS) of Nuclear Factor-κB(NF-κB). Such a peptide inhibits NF-κB signaling to the nucleus bycompetitive inhibition of cytoplasmic/nuclear translocation mechanism.Both isomers of MPS were able to deliver NLS to cytoplasm of murineendothelial LE II cells (FIG. 7A) and human erythroleukemia cells (FIG.7B), as evidenced by concentration-dependent inhibition of nuclearimport of NF-κB induced by proinflammatory agonists LPS (FIG. 7A) andTNF-α (FIG. 7B). Thus, intracellular delivery of functional peptides isnot dependent on chirality of MPS, indicating that a specific receptoror transporter protein is not involved. Moreover, MPS made of allD-amino acids renders this part of imported peptides resistant topeptidases.

EXAMPLE VII Inhibition of Inflammatory Skin Reaction by theCell-Permeable SN50 Peptide

Proinflammatory agents in contact with the skin cause localizedinflammatory reactions. For example, such a reaction can be elicited bybacterial lipopolysaccharide (Salmonella typhosa LPS 200 μg/ml insterile, pyrogen-free saline) which is applied first to rabbits as anintradermal injection (preparatory dose). To elicit localizedinflammatory reaction at the site of the first injection, a secondinjection of LPS (100 μg/kg body weight) is administered within 18-24hours intravenously into a rabbit ear vein. Subsequently, usually afterabout 4 hrs, a change at the site of initial skin injection is detected.It is manifested by a localized swelling due to increased vascularpermeability, redness due to vasodilation, and accumulation of whileblood cells and platelets in the skin blood vessels. This reaction,termed “localized Shwartzman reaction,” can be visualized by intravenousinjection of a biologic dye such as Evans' Blue. Thus, a positivereaction is manifested at the skin site of the first LPS injection as ablue area of inflammatory reaction. Intradermal application of thecell-permeable peptide SN50, prior to the second LPS injection, reducedlocalized skin inflammatory reaction as reflected by a greatlydiminished area of blue discoloration. When diluent alone (pyrogen-freesaline solution) was administered as a negative control, the localizedskin inflammatory reaction remained unchanged. Thus, the cell-permeablepeptide SN50, applied topically to the skin, can reduce localizedinflammatory reaction. This model or other known models of inflammatoryskin disease can be used to further study the efficacy of cell-permeablepeptides (for example, linear or cyclic peptides that contain an NF-κBNLS, e.g., SN50 or cSN50) and to identify analogs of these peptides thatinhibit inflammatory responses in skin. Such NF-κB NLS-based peptidescan be used to treat, prevent, or reduce the effects of inflammatorydiseases and conditions of the skin involving autoimmune or allergicresponses, for example, but not limited to, psoriasis, eczema, contactdermititis (for example, due to contact with poison ivy or poison oak,nickel, latex, environmental toxins, or bacterial or fungal infections,i.e., those causing “athletes foot” or “jock itch”). The peptides canalso be used to treat, prevent, or reduce the inflammatory effects ofchemical or thermal burns to the skin.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

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1. An isolated peptide comprising a cell membrane-permeable hydrophobicregion of a signal peptide and a cyclized NF-kB nuclear localizationsequence comprising the amino acid sequenceCys-Xaa-Xaa-Gln-Arg-Lys-Arg-Gln-Lys-Xaa-Xaa-Xaa-Cys (SEQ ID NO:14),wherein Xaa is any amino acid or is absent.
 2. The isolated peptide ofclaim 1, wherein the cyclized NF-kB nuclear localization sequencecomprises the amino acid sequenceCys-Tyr-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys (SEQ ID NO:15). 3.The isolated peptide of claim 1, wherein the isolated peptide comprisesthe amino acid sequence set forth in SEQ ID NO: 12.